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G L Archer

Publications and source records attributed to G L Archer.

At least 19 recordsLinked to original sources
PubMed1996

Characterization of IS1272, an insertion sequence-like element from Staphylococcus haemolyticus.

We have previously shown (G. L. Archer, D. M. Niemeyer, J. A. Thanassi, and M. J. Pucci, Antimicrob. Agents Chemother. 38:447-454, 1994) that some methicillin-resistant staphylococcal isolates contain a partial deletion of the genes (mecR1 and mecI) that regulate the transcription of the methicillin resistance structural gene (mecA). When a fragment of DNA inserted at the point of the mecR1 deletion was used as a probe, hybridization with multiple bands was detected for Staphylococcus haemolyticus genomic DNA. In the present study, DNA sequencing of four unique clones recovered from a lambda library of S. haemolyticus revealed identical 1,934-bp elements. Each element, designated IS1272, contained 16-bp terminal inverted repeats (sequence identity, 15 of 16 bp) and two open reading frames of 819 and 687 bp; there were no flanking target site duplications. Database searches yielded amino acid homology with proteins predicted to be encoded by open reading frames from a putative insertion sequence element from Enterococcus hirae. DNA probes from each end and the middle of IS1272 were hybridized with restriction endonuclease-digested genomic DNA from clinical S. haemolyticus, Staphylococcus epidermidis, and Staphylococcus aureus isolates. Each of the 20 or more copies of the element found in S. haemolyticus isolates was intact, and copies were found in most chromosomal SmaI fragments. S. aureus and S. epidermidis isolates contained mostly incomplete fragments of the element, and there were many more hybridizing fragments in methicillin-resistant than in methicillin-susceptible isolates. IS1272, which appears to be primarily resident in S. haemolyticus, has disseminated to multiple staphylococcal species and is prevalent in multiresistant isolates.

Amino Acid Sequence↗
PubMed1996

Identification and characterization of the origin of conjugative transfer (oriT) and a gene (nes) encoding a single-stranded endonuclease on the staphylococcal plasmid pGO1.

The genes mediating the conjugative transfer of the 52-kb staphylococcal plasmid pGO1 are within a 14.4-kb gene cluster designated trs. However, a clone containing trs alone cannot transfer independently and no candidate oriT has been found within or contiguous to trs. In this study, we identified a 1,987-bp open reading frame (ORF) 24 kb 3' and 13 kb 5' to trs that was essential for conjugative transfer: transposon insertions into the ORF abolished transfer and a plasmid containing the ORF could complement these transposon-inactivated pGO1 mutants for transfer. Analysis of the nucleotide sequence of this ORF revealed significant homology between the amino terminus of its predicted protein and those of several single-stranded endonucleases. In addition, a 12-bp DNA sequence located 100 bp 5' to the ORF's translational start site was identical to the oriT sequences of the conjugative or mobilizable plasmids RSF1010, pTF1, R1162, pSC101, and pIP501. The ability of the ORF, designated nes (for nicking enzyme of staphylococci), to generate a single-stranded nick at the oriT was demonstrated in Escherichia coli by alkaline gel and DNA sequence analysis of open circular plasmid DNA. Plasmids that could be converted to the open circular form by the presence of oriT and nes could also be mobilized at high frequency into Staphylococcus aureus recipients with a second plasmid containing only trs. We propose that the 14.4 kb of trs and the approximately 2.2 kb of the oriT-nes region, coupled with an origin of replication, make up the minimal staphylococcal conjugative replicon.

Amino Acid Sequence↗
PubMed1996

Role of mecA transcriptional regulation in the phenotypic expression of methicillin resistance in Staphylococcus aureus.

The gene required for methicillin resistance in staphylococci, mecA, encodes the low-affinity penicillin-binding protein 2a (PBP2a). Transcriptional regulation of mecA is accomplished in some isolates by mecR1 and mecI, cotranscribed chromosomal genes that encode a putative signal transducer and a transcriptional repressor, respectively. Two Staphylococcus aureus strains that have identical mecR1-mecI nucleotide sequences, BMS1 and N315P, both exhibit low-level, heterotypic expression of methicillin resistance and contain no beta-lactamase coregulatory sequences. mecR1-mecI was amplified from BMS1 by PCR and was shown to be functional on a high-copy-number plasmid when introduced into an S. aureus strain with a deleted mecR1-mecI locus. Cloned mecR1-mecI repressed phenotypic expression of methicillin resistance, mecA transcription and PBP2a production and mediated PBP2a induction in response to certain beta-lactam antibiotics. However, mecR1-mecI had different regulatory activities in its native chromosomal location in N315P compared with those in BMS1. Uninduced mecA transcription was markedly repressed in N315P, and mecI inactivation increased mecA transcription and PBP2a production 5- and 40-fold, respectively. Furthermore, the N315P phenotype changed from low-level, heterotypic resistance with intact mecI to high-level, homotypic resistance in strains with disrupted mecI. In contrast, uninduced BMS1 produced abundant mecA transcript and PBP2a, while the disruption of mecI had no effect on phenotype and little effect on mecA transcription or PBP2a production. Thus, mecI-mediated repression of mecA appears to be dysfunctional in BMS1 because of the presence or absence of additional regulatory cofactors. Furthermore, heterotypic resistance expression in this strain is independent of mecA transcriptional regulation.

Bacterial Proteins↗
PubMed1995

Characterization of a conjugative staphylococcal mupirocin resistance plasmid.

We studied conjugative plasmids encoding high-level mupirocin resistance. These plasmids were found in Staphylococcus aureus isolates from two geographic locations in the United States. Transfer genes on three mupirocin resistance plasmids with different restriction endonuclease profiles were indistinguishable by DNA hybridization from those on pG01, a conjugative aminoglycoside resistance plasmid representative of similar plasmids that are prevalent in the United States. One mupirocin resistance plasmid, pG0400 (34 kb), was smaller than pG01 (52 kb) because of the absence from pG0400 of DNA, found on pG01, that contained genes encoding resistance to aminoglycosides, trimethoprim, and quaternary ammonium compounds flanked by directly repeated copies of the insertion sequence (IS)-like element IS431-IS257. The plasmids pG0400 and pG01 were otherwise indistinguishable except for the presence in pG0400 of a 4.5-kb HinDIII fragment encoding mupirocin resistance. The added mupirocin resistance gene was flanked by two directly repeated copies of IS431/257. The nucleotide sequence of DNA contiguous to the outside of the IS elements, as well as those of the elements themselves, was identical in both pG01 and pG0400, and there were no target site duplications flanking either copy of the element. We conclude that the mupirocin resistance gene was added to an existing conjugative plasmid in conjunction with the deletion of other resistance genes by recombination at IS elements. The construction of conjugative plasmids carrying a mupirocin resistance gene may be a model for the mobility of other resistance genes newly acquired by staphylococci.

Base Sequence↗
PubMed1994

Origin and evolution of DNA associated with resistance to methicillin in staphylococci.

The gene mediating resistance to methicillin in staphylococci (mecA) and its flanking sequences (mec DNA) make up a chromosomal DNA locus that is unique to methicillin-resistant bacteria; no equivalent locus exists in methicillin-susceptible cells. The origin of mec DNA is not known, but evidence supports horizontal transfer of mec DNA between different staphylococcal species and of the mecA gene between different Gram-positive genera.

Chromosome Mapping↗
PubMed1994

Coagulase-negative staphylococci: pathogens associated with medical progress.

Coagulase-negative staphylococcal bacteremia and infections of prosthetic medical devices have become major clinical problems. Efforts to differentiate contaminating from infecting isolates consume the time of microbiology laboratory personnel; decisions over when and with what to institute therapy for multiresistant isolates consume the energy of clinicians; and the need to institute expensive parenteral antimicrobial therapy consumes the hospital pharmacy budget. It is clear that the increased incidence of coagulase-negative staphylococcal infections is the result of medical progress and is due to the use of invasive and indwelling medical devices. Multiresistant organisms have evolved that will survive in the presence of antimicrobial agents designed to eradicate more traditional pathogens. They have an ecological niche on human skin from which they are difficult to eradicate, and they have adapted themselves to survive on inert devices designed to persist indefinitely in the human body. Since it is likely that the use of prosthetic medical devices will continue to increase, we need to device innovative strategies for the diagnosis, treatment, and prevention of infections of these indwelling foreign bodies. Studies that will address these issues should be a major goal of future research on hospital-acquired infections.

Bacteremia↗
PubMed1994

Dissemination among staphylococci of DNA sequences associated with methicillin resistance.

DNA probes consisting of pUC19 containing cloned Staphylococcus aureus chromosomal fragments were constructed from two methicillin-resistant S. aureus strains with different DNA sequences 5' to mecA, the gene that mediates methicillin resistance. The probe from one strain, BMS1, contained a portion of the regulatory sequences (the terminal 641 bp of mecR1 and all of mecI) associated with the induction and repression of mecA transcription (pGO195). The second probe, from strain COL (pGO198), contained DNA not found in strain BMS1. This DNA was within the sequences added at the site of a mecR1 deletion. Genomic digests of 14 S. aureus isolates recovered between 1961 and 1969 all hybridized with pGO198. In contrast, 78% (36 of 46) of the S. aureus organisms isolated since 1988 hybridized with pGO195 but not with pGO198; the remainder hybridized with pGO198. No S. aureus isolates hybridized with both probes. Staphylococcus epidermidis digests hybridized with pGO198 (46%), pGO195 (14%), or both probes (35%); all 20 Staphylococcus haemolyticus isolates hybridized with pGO198. The restriction fragment length polymorphism patterns of all pGO198-hybridizing regions in S. aureus were identical to those in strain COL. In addition, the mecR1 deletion junction nucleotide sequences of eight S. aureus and six S. epidermidis isolates were identical. However, 21 of 23 S. epidermidis and all 20 S. haemolyticus isolates had from 5 to more than 20 additional chromosomal bands that hybridized with pGO198; none of 21 S. aureus isolates had additional hybridizing bands. These data suggest that the additional DNA responsible for the mecR1 deletion was part of a repetitive, and possibly mobile, element resident in coagulase-negative staphylococci but not in S. aureus. These data also support a hypothesis that the deletion event occurred in a coagulase-negative staphylococcus with subsequent acquisition of the interrupted sequences by S. aureus.

Base Sequence↗
PubMed1994

Transcriptional regulation by TrsN of conjugative transfer genes on staphylococcal plasmid pGO1.

The major conjugative transfer gene cluster of staphylococcal plasmid pGO1 (trs) consists of 13 open reading frames (trsA to trsM) transcribed from one DNA strand and a single 189-bp open reading frame (trsN) within the first 348 bp of trs that is transcribed divergently. Promoter regions for trsN and trsA partially overlap. TrsN, a 7,181-Da protein, was purified as a fusion to glutathione S-transferase and found to have DNA-binding activity. Increasing concentrations of the fusion protein progressively retarded the gel migration of PCR-generated DNA fragments containing predicted promoters 5' to trsL, trsA, and trsN. The target sequences contained areas of identity, including regions of dyad symmetry, that were protected in DNase I footprinting studies. The binding of TrsN to its trsL target was required for this target DNA to be stably introduced into Staphylococcus aureus on a high-copy-number vector. Provision of excess TrsN from this high-copy-number vector in S. aureus decreased beta-galactosidase activity from a trsL-lacZ transcriptional fusion and decreased pGO1 conjugation frequency. Conversely, both transcription and conjugation increased in the presence of excess trsL target. We propose that TrsN negatively regulates the transcription of genes essential for conjugative transfer by binding to regions 5' to their translational start sites.

Amino Acid Sequence↗
PubMed1993

DNA sequence and units of transcription of the conjugative transfer gene complex (trs) of Staphylococcus aureus plasmid pGO1.

The conjugative transfer genes of 52-kb staphylococcal R plasmid pGO1 were localized to a single BglII restriction fragment and cloned in Escherichia coli. Sequence analysis of the 13,612-base transfer region, designated trs, identified 14 intact open reading frames (ORFs), 13 of which were transcribed in the same direction. Each ORF identified was preceded by a typical staphylococcal ribosomal binding sequence, and 10 of the 14 proteins predicted to be encoded by these ORFs were seen when an E. coli in vitro transcription-translation system was used. Functional transcription units were identified in a Staphylococcus aureus host by complementation of Tn917 inserts that abolished transfer and by Northern (RNA) blot analysis of pGO1 mRNA transcripts. These studies identified three complementation groups (trsA through trsC, trsD through trsK, and trsL-trsM) and four mRNA transcripts (trsA through trsC [1.8 kb], trsA-trsB [1.3 kb], trsL-trsM [1.5 kb], and trsN [400 bases]). No definite mRNA transcript was seen for the largest complementation group, trsD through trsK (10 kb). Comparison of predicted trs-encoded amino acid sequences to those in the data base showed 20% identity of trsK to three related genes necessary for conjugative transfer of plasmids in gram-negative species and 32% identity of trsC to a gene required for conjugative mobilization of plasmid pC221 from staphylococci.

Amino Acid Sequence↗
PubMed1992

Mobilization of recombinant plasmids from Staphylococcus aureus into coagulase negative Staphylococcus species.

pC221, a small nonconjugative staphylococcal plasmid, can be mobilized between staphylococci by pG01, a larger conjugative plasmid. pC221 carries the two transacting genes, mobA and mobB, which are needed for its mobilization. The products of these genes create a site-specific single-stranded nick (mobA) and then facilitate DNA transfer (mobB). Several useful Escherichia coli-staphylococcal shuttle plasmids containing the cloned single-stranded nick site were created and successfully mobilized into Staphylococcus aureus and two coagulase-negative staphylococci, S. epidermidis and S. saprophyticus, by providing mob genes (pC221) and conjugative transfer genes (pG01) in trans in the donor. These vectors may offer a genetic system for the introduction of recombinant plasmids into coagulase negative staphylococci.

Bacterial Proteins↗
PubMed1992

Hemagglutination and adherence to plastic by Staphylococcus epidermidis.

Staphylococcus epidermidis is an important nosocomial pathogen responsible for intravenous catheter-related bacteremia and infections of other prosthetic medical devices. We found that the ability of S. epidermidis to hemagglutinate erythrocytes correlated with the adherence of bacteria to plastic and to intravenous catheters. S. epidermidis isolates responsible for prosthetic-valve endocarditis (n = 61) and isolates from intravenous catheters (n = 59) were significantly more likely to cause hemagglutination than isolates from the skin of preoperative cardiac surgery patients (n = 19) (P = 0.027). S. epidermidis isolates (n = 23) recovered from the skin of patients 7 to 10 days after cardiac surgery were significantly more likely to exhibit hemagglutination than the preoperative isolates (P = 0.015). By a quantitative adherence assay, we also observed that the hemagglutination titer and number of species of erythrocytes agglutinated correlated directly with adherence to polystyrene (P less than 0.001). In addition, hemagglutinating isolates were significantly more likely to be recovered in high number from intravenous catheters when semiquantitative catheter culture techniques were used (P less than 0.001). We speculate that hemagglutinin(s) either plays a direct role in adherence to polymers and thus prosthetic-device infection or serves as an easily demonstrable marker for adherence-prone isolates.

Animals↗
PubMed1992

Colonization of the female genital tract with Staphylococcus saprophyticus.

The prevalence of colonization by Staphylococcus saprophyticus of the urogenital tracts of 276 women from an outpatient gynecology practice was determined by using selective and enrichment culture techniques. Nineteen subjects (6.9%) were found to be colonized by S. saprophyticus. The rectum was the most frequent site of colonization and was responsible for 40% of the isolates; this was followed in decreasing order by the urethra, urine, and cervix. Women colonized by S. saprophyticus were more likely to have experienced a urinary tract infection in the previous 12 months (P = 0.058; odds ratio, 2.844; 95% confidence interval, 1.054 to 7.671). Patients colonized by S. saprophyticus tended to have had their menstrual periods more recently (P = 0.066), experienced sexual intercourse more recently (P = 0.168), and had a recent or concurrent diagnosis of vaginal candidiasis (P = 0.111; odds ratio, 2.393; 95% confidence interval, 0.877 to 6.528). A seasonal variation in colonization was observed, with colonization most likely occurring during the summer and fall. Follow-up for an average of 6.75 months failed to document any colonized woman progressing to symptomatic urinary tract infection. In addition, 21 women colonized by non-S. saprophyticus, novobiocin-resistant, coagulase-negative staphylococci were identified and characterized.

Adult↗
PubMed1991

Conjugative transfer genes in staphylococcal isolates from the United States.

Staphylococcus aureus and coagulase-negative staphylococcal isolates from various geographic areas in the United States were examined by using a conjugative transfer gene DNA probe in dot-blot hybridization assays. Of 175 S. aureus isolates, 47 (27%) hybridized with the probe, while 24 of 208 (11.5%) coagulase-negative staphylococci hybridized. However, among methicillin-resistant S. aureus 52% (45 of 89) were probe positive while only 2% (2 of 86) of methicillin-susceptible S. aureus were probe positive. In contrast, 12.5% (22 of 176) of methicillin-resistant and 6% (2 of 32) of methicillin-susceptible coagulase-negative staphylococci contained transfer genes. All but one of the staphylococci containing transfer genes were resistant to gentamicin; 91.5% of S. aureus and 65% of coagulase-negative staphylococci containing transfer genes transferred gentamicin resistance to a S. aureus recipient. Of the 12 isolates that hybridized with the probe but did not transfer resistance, 10 (6 coagulase-negative staphylococci and 4 S. aureus) carried both gentamicin resistance and conjugative transfer genes on the same plasmid. Of these 10, 6 contained plasmid target fragments of sizes different from that of the probe, suggesting additions or deletions of DNA essential for transfer, while in 4 no such alterations could be detected. In two coagulase-negative staphylococci the entire transfer region was apparently integrated into the chromosome. Thus, staphylococci carrying conjugative transfer genes are widely disseminated in the United States and are usually found in multiresistant isolates on plasmids that also encode gentamicin resistance.

Anti-Bacterial Agents↗
PubMed1990

Detection of methicillin resistance in staphylococci by using a DNA probe.

A DNA probe derived from the PBP 2a gene of the methicillin-resistant Staphylococcus aureus COL was compared with phenotypic microbiologic tests for its ability to identify methicillin-resistant and -susceptible staphylococci. Lysates were applied to nitrocellulose with a dot blot apparatus. Isolates tested were both S. aureus and coagulase-negative staphylococci that had been recovered from a variety of geographic and clinical sources. When compared with a spread plate phenotypic test, the DNA probe gave sensitivity, specificity, and predictive values for both positive and negative tests of 100% for 204 S. aureus isolates (103 positive, 101 negative) and 99, 95, 99, and 95%, respectively, for 249 coagulase-negative staphylococci (210 positive, 39 negative). The probe was more sensitive than broth microdilution and more specific than agar dilution in identifying methicillin-resistant and -susceptible coagulase-negative staphylococci; all tests were equally accurate in identifying the methicillin susceptibility of S. aureus. DNA probe analysis for determining the methicillin susceptibility of staphylococci was rapid, easily interpretable, and equally accurate with radioactive and nonradioactive probes, and it gave results equivalent to the most sensitive microbiologic test for all staphylococcus species studied.

Bacterial Proteins↗
PubMed1989

Antimicrobial resistance in nosocomial isolates of Staphylococcus haemolyticus.

Staphylococcus haemolyticus is frequently cultured from hospitalized patients and is characterized by resistance to multiple antimicrobial agents. We found that S. haemolyticus represented 70 of 524 (13%) coagulase-negative staphylococcal isolates identified by the clinical microbiology laboratories of two hospitals over 2 months. S. haemolyticus isolates were recovered from wounds (44%), urine (26%), blood (10%), and other sources (20%). All S. haemolyticus isolates were tested for susceptibility to six antimicrobial agents; 77% were resistant to three or more agents, and 41% were resistant to five or six agents. In addition, among 47 multiply resistant isolates, high MICs (greater than or equal to 6.25 micrograms/ml) of vancomycin (62% of isolates) and teicoplanin (91% of isolates) were found. DNA probes which were derived from S. epidermidis or S. aureus and which contained sequences associated with resistance to antimicrobial agents were used to detect specific genes in the total cellular and plasmid DNAs of 10 resistant S. haemolyticus isolates. Resistance gene probes and the numbers of resistant isolates hybridizing were as follows: methicillin, 10 of 10; gentamicin, 9 of 10; erythromycin, 7 of 10; and trimethoprim, 0 of 10. Genes for resistance to methicillin were found only in chromosomal locations, genes for resistance to gentamicin were found in both chromosomal and plasmid locations, and genes for resistance to erythromycin were found in plasmid locations only. With the exception of trimethoprim resistance determinants, similar genes were found among concurrently isolated multiply resistant S. epidermidis isolates from our hospitals. S. haemolyticus is a potentially important nosocomial species which readily acquires antimicrobial resistance genes and which shares, to some extent, in a common gene pool with S. epidermidis.

Anti-Bacterial Agents↗